1. Field of the Invention
This invention is related to interfacing liquid chromatography to mass spectrometry. More specifically it is related to interfacing liquid nano- and micro-capillary chromatography to mass spectrometry using electrospray ionization.
2. Description of the Related Art
It is well recognized that conducting liquid chromatography (LC) at elevated temperatures (typically 30-80° C.) results in beneficial effects, such as improvements in peak shape, selectivity, retention time stability, chromatographic resolution, and speed of analysis. [Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development, 2nd ed.; Wiley & Sons: New York, 1997; Dolan, J. W. J. Chromatogr., A 2002, 965 (1-2), 195-205.5,6]. Temperature affects several physical parameters in liquid chromatography such as retention factor of analytes, analyte diffusion, and mobile phase viscosity.
High Performance Liquid Chromatographic (HPLC) or Ultra Performance Liquid Chromatography (UPLC) separations performed in capillary as well as in large-scale HPLC columns those with the internal diameter (i.d.) larger than 0.5 mm, at elevated temperatures have also other advantages such as reducing carryover, lowering column pressure, and increasing speed of the analysis [Rosés M, Subirats X, Bosch E., J. Chromatogr A. 2009 Mar. 6; 1216(10):1756-75].
The use of mass spectrometry (MS) as a detector of compounds separated by LC substantially increased the applicability of the chromatography as an analytical method. Typically, an electrospray ionization (ESI) technique is used to ionize the analyte molecules containing in the eluent before directing the analyte ions to mass spectrometer (MS) or ion mobility spectrometer (IMS) for chemical analysis. In electrospray, the liquid sample is sprayed using a silica or metal capillary (an ESI tip) by the action of high voltage applied to the tip, column, HPLC eluent, or liquid sample to be sprayed. After generation, the charged liquid micro-droplets start to evaporate. The analyte ions are formed from charged micro-droplets in a process of solvent evaporation that is accompanied by a multiple droplet division due to Coulomb repulsion of surface charges. After the droplet diameter shrinks to some critical size, the analyte ions are produced in the eventual process of field emission.
An integrated LC column typically has a silica capillary that is ended with an ESI tip made from the same silica capillary by pulling the capillary end at high temperature to make its internal and outer diameter smaller, although the ESI tip can be fabricated separately. This integrated column/ESI tip assembly is packed with chromatographic media starting from the open end of the column to its tip. (When the tip is a separate body made of narrow internal diameter fused silica, metal, glass and other materials, it is typically not packed.) The integrated column is connected via union to the supply capillary with a flow of a mobile phase from an HPLC pump. Mobile phases are typically mixtures of organic and aqueous solvents (e.g., water mixed with acetonitrile) with the flow rate typically in the range of 100-1000 nL/min. This so-called nano-flow regime gives the term of nano-LC for the columns operating at nL/min flow rates [Chen G, Pramanik BN, LC-MS for protein characterization: current capabilities and future trends. Expert Rev Proteomics. 2008; 5(3): 435-44.] Similarly, micro-capillary LC (or micro-LC) corresponds to the flow rates in the μL/min range. Such low flow rates typically require the use of ESI tips with narrow droplet emitting channels.
Compared to capillary and large-scale HPLC columns, operating integrated nano-LC/ESI at the elevated temperatures is not straightforward. As an example, the data for nano-LC column with a compact heater recently commercialized by Phoenix S&T (Chester, Pa., USA) indicate that the use of elevated temperatures broadens the LC peaks for individual peptides (they are up to 2 times wider at 50° C. compared to those at 20° C.).
The entire contents of the references listed above are incorporated herein in their entirety by reference.